In the last granting period, we were successful in developing a genetic approach to rescue the deficiency in ATP synthesis in cellular models of maternally-inherited Leigh syndrome (MILS) due to mutations in mitochondrial DNA (mtDNA)-encoded ATPase 6, using a strategy called "allotopic expression." We also began work on developing a pharmacological approach to treat these disorders, based on our finding that treatment of heteroplasmic cells containing the MILS mutation with an ATPase-specific inhibitor (oligomycin) in medium containing galactose resulted in a rapid and stable shift in heteroplasmy in favor of wild-type mtDNAs, with a concomitant improvement in mitochondrial function. Importantly, we have now shown that this type of heteroplasmic shifting" strategy can be applied to mitochondrial deficiencies in other respiratory complexes, including those in Kearns-Sayre syndrome (KSS), which is characterized by large-scale deletions of mtDNA (delta-mtDNAs). Furthermore, instead of using the relatively toxic galactose/oligomycin medium, we were able to use a relatively non-toxic medum containing ketone bodies (i.e. acetoacetate and/or beta-hydroxybutyrate) as the sole carbon source in order to select for function and reduce significantly the amount of delta-mtDNAs in a replicating cell system (i.e. cytoplasmic hybrid [cybrid] cells). We now propose to follow up on this promising approach to therapy in three ways. First, we will ask if ketogenic media can select for wild-type function, and mtDNAs, in cells containing other pathogenic mtDNA mutations, in order to see the degree to which this treatment strategy can be generalized, and will also ask if ketogenic selection can work in the presence of other substrates (fatty acids, amino acids) as well as in the presence of low levels of glucose, so as to mimic the clinical situation more closely. Second, we will ask if we can shift heteroplasmy in a terminally-differentiated, non-replicating, model system, namely myotubes, in order to nail down the issue of inter- vs -intra-cellular selection against mutated mtDNAs in ketogenic medium. Finally, we will perform fluorescent in situ hybridization in KSS cells to determine in a more mechanistic fashion how mtDNAs segregate in both dividing (i.e. cybrid) and non-dividing (i.e. myotube) cells, during growth in both glucose ("rich" medium) and in ketones ("selective" medium). These experiments are designed to provide a more mechanistic underpinning for our observation of ketone-mediated selection against mutated mtDNAs, as a prelude to using a ketogenic-based protocol to treat patients with heteroplasmic pathogenic mutations in mtDNA.